Global regulatory systems modulate the expression of numerous genes located throughout the genome, permitting the overall physiological, metabolic, and developmental status of the organism to respond rapidly and sometimes dramatically to changes in the environment. The term “global regulator” describes a relatively small number of genes whose products have a wide-ranging influence on the state of the cell. One function of these regulators is to code for products that bind promoter elements, such as enhancers or silencers, of the gene whose expression they influence; other regulators function by activating or inactivating a cascading series of cellular reactions. The potential to create powerful regulatory systems in microbial strains using these regulators is only now beginning to be appreciated.
The health benefits associated with polyunsaturated fatty acids [“PUFAs’] have been well documented. As a result, considerable research has been directed toward production of large-scale quantities of PUFAs by: 1) cultivation of microbial organisms, such as heterotrophic diatoms Cyclotella sp. and Nitzschia sp.; Pseudomonas, Alteromonas or Shewanella species; filamentous fungi of the genus Pythium; or Mortierella elongata, M. exigua or M. hygrophila, that natively produce the fatty acid of choice; and 2) discovery of fatty acid desaturase and elongase genes that permit synthesis of fatty acids and subsequent introduction of these genes into organisms that do not natively produce ω-3/ω-6 PUFAs via genetic engineering methods. However, commercial exploitation of this work has been limited because of limited production of the preferred ω-3/ω-6 PUFAs and/or inability to substantially improve the yield of oil/control the characteristics of the oil composition produced.
Commonly owned U.S. Pat. No. 7,238,482 describes the use of oleaginous yeast Yarrowia lipolytica as a production host for the production of PUFAs. Oleaginous yeast are defined as those yeast that are naturally capable of oil synthesis and accumulation, where greater than 25% of the cellular dry weight is typical. Optimization of the production host has been described in the art (see for example Intl. App. Pub. No. WO 2006/033723, U.S. Pat. Appl. Pub. No. 2006-0094092, U.S. Pat. Appl. Pub. No. 2006-0115881, U.S. Pat. Appl. Pub. No. 2006-0110806 and U.S. Pat. Appl. Pub. No. 2009-0093543-A1). The recombinant strains described therein comprise various chimeric genes expressing multiple copies of heterologous desaturases and elongases, and optionally comprise various native desaturase, acyltransferase and perioxisome biogenesis protein knockouts to enable PUFA synthesis and accumulation.
Further optimization of the host cell is needed for commercial production of PUFAs. The inventors were interested in identifying a global regulator in oleaginous organisms that would uncouple the process of lipid biosynthesis from the oleaginous stage of growth. Such a regulatory element would be extremely desirable because it would possess broad specificity for the activation and/or repression of secondary metabolite genes while providing strains capable of otherwise normal or near-normal development and growth.
It has been found that reduction in activity of the heterotrimeric SNF1 protein kinase results in increased accumulation of lipids in Yarrowia lipolytica. Despite numerous previous studies concerning the heterotrimeric SNF1 protein kinase network, many details concerning the cellular role of SNF1 protein kinase and its regulation are still not fully understood and remain to be elucidated. Also, previous studies of SNF1 knockouts have not been performed in an oleaginous organism. Although the AMPK/SNF1 kinase family is highly conserved throughout eukaryotes and required for the maintenance of cellular energy homeostasis, its specific regulatory mode may be different among different organisms.
This appears to be the first discovery of a generalized mechanism wherein reduction in the activity of the heterotrimeric SNF1 protein kinase leads to the surprising discovery of increased lipid biosynthesis, resulting in constitutive oleaginy.